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PubMed2005

Proteome analyses of Staphylococcus aureus in growing and non-growing cells: a physiological approach.

Staphylococcus aureus is a versatile human pathogen causing a wide variety of diseases ranging from wound infection to endocarditis, osteomyelitis, and sepsis. In order to investigate this pathogen, we sought to analyze the cytoplasmic proteome of S. aureus COL by using two different approaches: two-dimensional (2D) gel analyses combined with matrix-assisted laser ionization-time of flight mass spectrometry and a gel-free system using multidimensional liquid chromatography followed by mass spectrometry. By combining both analyses we identified 1123 cytoplasmic proteins that represent two-thirds of the cytoplasmic proteome of the organism. With our standard 2D gel setup (pI 4-7) we identified 473 proteins that cover about 40% of the cytoplasmic proteome predicted for this proteomic window. The identified proteins belong to a variety of cellular functions ranging from the transcriptional and translational machinery, tricarboxylic acid cycle (TCC), glycolysis, and fermentation pathways to biosynthetic pathways of nucleotides, fatty acids, and cell wall components. While most of the metabolic pathways predicted for S. aureus were covered by this gel-based proteomics 650 additional proteins were identified by the gel-free approach, among them alkaline or hydrophobic proteins. In our work, we established a master 2D gel that enabled us to study the regulation of core carbon metabolism in S. aureus cells grown in a complex medium. Our comparison of the protein pattern of exponentially growing cells with that of stationary-phase cells revealed a higher amount of enzymes involved in protein synthesis, transcription, and glycolysis in exponentially growing cells. In contrast, enzymes of the TCC and gluconeogenesis are increased at the stationary phase. With this comprehensive proteome map we have an essential tool for a better understanding of cell physiology of the human pathogen, S. aureus.

Adaptation, Physiological↗

PubMed2006

Microbial genomics for the improvement of natural product discovery.

The quest for the discovery of novel natural products has entered a new chapter with the enormous wealth of genetic data that is now available. This information has been exploited by using whole-genome sequence mining to uncover cryptic pathways, or biosynthetic pathways for previously undetected metabolites. Alternatively, using known paradigms for secondary metabolite biosynthesis, genetic information has been 'fished out' of DNA libraries resulting in the discovery of new natural products and isolation of gene clusters for known metabolites. Novel natural products have been discovered by expressing genetic data from uncultured organisms or difficult-to-manipulate strains in heterologous hosts. Furthermore, improvements in heterologous expression have not only helped to identify gene clusters but have also made it easier to manipulate these genes in order to generate new compounds. Finally, and perhaps the most crucial aspect of the efficient and prosperous use of the abundance of genetic information, novel enzyme chemistry continues to be discovered, which has aided our understanding of how natural products are biosynthesized de novo, and enabled us to rework the current paradigms for natural product biosynthesis.

Bacteria↗

PubMed2006

In vitro evidence for the dual function of Alg2 and Alg11: essential mannosyltransferases in N-linked glycoprotein biosynthesis.

The biosynthesis of asparagine-linked glycoproteins utilizes a dolichylpyrophosphate-linked glycosyl donor (Dol-PP-GlcNAc(2)Man(9)Glc(3)), which is assembled by the series of membrane-bound glycosyltransferases that comprise the dolichol pathway. This biosynthetic pathway is highly conserved throughout eukaryotic evolution. While complementary genetic and bioinformatic approaches have enabled identification of most of the dolichol pathway enzymes in Saccharomyces cerevisiae, the roles of two of the mannosyltransferases in the pathway, Alg2 and Alg11, have remained ambiguous because these enzymes appear to catalyze only two of the remaining four unannotated transformations. To address this issue, a biochemical approach was taken using recombinant Alg2 and Alg11 from S. cerevisiae and defined dolichylpyrophosphate-linked substrates. A cell-membrane fraction isolated from Escherichia coli overexpressing thioredoxin-tagged Alg2 was used to demonstrate that this enzyme actually carries out an alpha1,3-mannosylation, followed by an alpha1,6-mannosylation, to form the first branched pentasaccharide intermediate of the pathway. Then, using thioredoxin-tagged Alg2 for the chemoenzymatic synthesis of the dolichylpyrophosphate pentasaccharide, it was thus possible to define the biochemical function of Alg11, which is to catalyze the next two sequential alpha1,2-mannosylations. The elucidation of the dual function of each of these enzymes thus completes the identification of the entire ensemble of glycosyltransferases that comprise the dolichol pathway.

Asparagine↗

PubMed2003

A reaction center-light-harvesting 1 complex (RC-LH1) from a Rhodospirillum rubrum mutant with altered esterifying pigments: characterization by optical spectroscopy and cryo-electron microscopy.

Introduction of the bchP gene from Rhodobacter sphaeroides encoding geranylgeranyl reductase into Rhodospirillum rubrum alters the esterification of the bacteriochlorophylls so that phytol is used instead of geranylgeraniol. The resulting transconjugant strain of Rs. rubrum grows photosynthetically, showing that phytolated Bchla can substitute for the native pigment in both the reaction center (RC) and the light-harvesting 1 (LH1) complexes. This genetic manipulation perturbs the native carotenoid biosynthetic pathway; several biosynthetic intermediates are assembled into the core complex and are capable of energy transfer to the bacteriochlorophylls. RC-LH1 complexes containing phytolated Bchla were analyzed by low temperature absorption and fluorescence spectroscopy and circular dichroism. These show that phytolated Bchls can assemble in vivo into the photosynthetic apparatus of Rs. rubrum and that the newly introduced phytol tail provokes small perturbations to the Bchls within their binding sites in the LH1 complex. The RC-LH1 core complex was purified from membranes and reconstituted into well ordered two-dimensional crystals with a p4212 space group. A projection map calculated to 9 A shows clearly that the LH1 ring from the mutant is composed of 16 subunits that surround the reaction center and that the diameter of this complex is in close agreement with that of the wild-type LH1 complex.

Bacteriochlorophylls↗

PubMedDate not supplied

The ins and outs of ring-cleaving dioxygenases.

Ring-cleaving dioxygenases catalyze the oxygenolytic fission of catecholic compounds, a critical step in the aerobic degradation of aromatic compounds by bacteria. Two classes of these enzymes have been identified, based on the mode of ring cleavage: intradiol dioxygenases utilize non-heme Fe(III) to cleave the aromatic nucleus ortho to the hydroxyl substituents; and extradiol dioxygenases utilize non-heme Fe(II) or other divalent metal ions to cleave the aromatic nucleus meta to the hydroxyl substituents. Recent genomic, structural, spectroscopic, and kinetic studies have increased our understanding of the distribution, evolution, and mechanisms of these enzymes. Overall, extradiol dioxygenases appear to be more versatile than their intradiol counterparts. Thus, the former cleave a wider variety of substrates, have evolved on a larger number of structural scaffolds, and occur in a wider variety of pathways, including biosynthetic pathways and pathways that degrade non-aromatic compounds. The catalytic mechanisms of the two enzymes proceed via similar iron-alkylperoxo intermediates. The ability of extradiol enzymes to act on a variety of non-catecholic compounds is consistent with proposed differences in the breakdown of this iron-alkylperoxo intermediate in the two enzymes, involving alkenyl migration in extradiol enzymes and acyl migration in intradiol enzymes. Nevertheless, despite recent advances in our understanding of these fascinating enzymes, the major determinant of the mode of ring cleavage remains unknown.

Bacterial Proteins↗

PubMed2004

Quantifying modularity in the evolution of biomolecular systems.

Functional modules are considered the primary building blocks of biomolecular systems. Here we study to what extent functional modules behave cohesively across genomes:That is, are functional modules also evolutionary modules? We probe this question by analyzing for a large collection of functional modules the phyletic patterns of their genes across 110 genomes. The majority of functional modules display limited evolutionary modularity. This result confirms certain comparative genome analyses, but is in contrast to implicit assumptions in the systems analysis of functional genomics data. We show that this apparent interspecies flexibility in the organization of functional modules depends more on functional differentiation within orthologous groups of genes, than on noise in the functional module definitions. When filtering out these sources of nonmodularity, even though very few functional modules behave perfectly modular in evolution, about half behave at least significantly more modular than a random set of genes. There are substantial differences in the evolutionary modularity between individual functional modules as well as between collections of functional modules, partly corresponding to conceptual differences in the functional module definition, which make comparisons between functional module collections biologically difficult to interpret. Analysis within one collection does not suffer from such differences, and we show that within the EcoCyc metabolic pathway database, biosynthetic pathways are evolutionarily more modular than catabolic pathways.

Computational Biology↗

PubMed2004

Structural similarity of YbeD protein from Escherichia coli to allosteric regulatory domains.

Lipoic acid is an essential prosthetic group in several metabolic pathways. The biosynthetic pathway of protein lipoylation in Escherichia coli involves gene products of the lip operon. YbeD is a conserved bacterial protein located in the dacA-lipB intergenic region. Here, we report the nuclear magnetic resonance structure of YbeD from E. coli. The structure includes a beta alpha beta beta alpha beta fold with two alpha-helices on one side of a four-strand antiparallel beta-sheet. The beta 2-beta 3 loop shows the highest sequence conservation and is likely functionally important. The beta-sheet surface contains a patch of conserved hydrophobic residues, suggesting a role in protein-protein interactions. YbeD shows striking structural homology to the regulatory domain from d-3-phosphoglycerate dehydrogenase, hinting at a role in the allosteric regulation of lipoic acid biosynthesis or the glycine cleavage system.

Allosteric Regulation↗

PubMed2000

Edward P. Abraham, cell-free systems and the fungal biosynthesis of beta-lactams.

Today much is known about the biology of penicillin and cephalosporin production by fungi including the pathways, the biosynthetic enzymes including some crystal structures, the genes and their cloning, expression, sequencing and chromosomal locations, the regulation of the genes and enzymes and even some intelligent guesses about their evolutionary relationships. The key breakthrough that led to rapid progress in these areas was the subcellular work done by EDWARD P. ABRAHAM and his Oxford colleagues in the early 1970s. With his advice and encouragement, my laboratory was able to prepare reliably active soluble cell-free preparations which were instrumental in elucidation of the biosynthetic pathways in fungi (and also in bacteria) by laboratories throughout the world.

Anti-Bacterial Agents↗

PubMed1977

Clinical relevance of polyamines as biochemical markers of tumor kinetics.

The polyamines, spermidine and spermine, and their diamine precursor, putrescine, constitute a unidirectional biosynthetic pathway whose biosynthetic enzymes and accumulation patterns appear to play important roles in the regulation of growth processes. Concentrations of these compounds in physiological fluids are low or undetectable under normal conditions, are elevated in patients with metastatic cancer, and are thought to reflect growth (putrescine concentrations) and cell turnover (spermidine concentrations) of the organism. Cancers are a broad spectrum of diseases in which there are altered growth fractions and cell-turnover fractions, and therefore cancer chemotherapeutic agents have been developed to take tumor kinetics into account. Because changes in polyamines in physiological fluids reflect cell kinetics, this review compiles evidence of their efficacy as biochemical markers of cancer and suggests their possible usefulness to clinicians in rapidly assessing tumor response to chemotherapy or to multimodality therapy.

Animals↗

PubMed1998

Effects of nucleic acid inhibitors on the development of Angiostrongylus costaricensis in vitro.

Nucleic acid biosynthesis of Angiostrongylus costaricensis was examined with various inhibitors; aminopterin (inhibitor of purine and pyrimidine de novo biosynthetic pathways), 8-azaguanine (specific inhibitor of purine salvage pathway) and PALA (specific inhibitor of pyrimidine de novo biosynthetic pathway) were applied in in vitro culture developing from the third stage larvae to young adult in chemically defined medium. It was suggested that A. costaricensis possessed functional purine and pyrimidine de novo biosynthetic pathways and also that they could utilize exogenous sources of purines and pyrimidines by salvage pathways for their development.

Aminopterin↗

PubMed2003

Neuropeptide conversion to bioactive fragments--an important pathway in neuromodulation.

Biosynthetic pathways for the formation of neuroactive peptides and the processes for their inactivation include several enzymatic steps. In addition to enzymatic processing and degradation, several neuropeptides have been shown to undergo enzymatic conversion to fragments with retained or modified biological activity. This has most clearly been demonstrated for e.g. opioid peptides, tachykinins, calcitonin gene-related peptide (CGRP) as well as for peptides belonging to the renin-angiotensin system. Sometimes the released fragment shares the activity of the parent compound. However, in many cases the conversion reaction is linked to a change in the receptor activation profile, i.e. the generated fragment acts on and stimulates a receptor not recognized by the parent peptide. This review will describe the characteristics of certain neuropeptide fragments having the ability to modify the biological action of the peptide from which they are derived. Focus will be directed to the tachykinins, the opioid peptides, angiotensins as well as to CGRP, bradykinin and nociceptin. The kappa opioid receptor selective opioid peptide, dynorphin, recognized for its ability to produce dysphoria, is converted to the delta opioid receptor agonist Leu-enkephalin, with euphoric properties. The tachykinins, typified by substance P (SP), is converted to the bioactive fragment SP(1-7), a heptapeptide mimicking some but opposing other effects of the parent peptide. The bioactive angiotensin II, known to bind to and stimulate the AT-1 and AT-2 receptors, is converted to angiotensin IV (i.e. angiotensin 3-8) with preference for the AT-4 sites or to angiotensin (1-7), not recognized by any of these receptors. Both angiotensin IV and angiotensin (1-7) are biologically active. For example angiotensin (1-7) retains some of the actions ascribed for angiotensin II but is shown to counteract others. Thus, it is obvious that the activity of many neuroactive peptides is modulated by bioactive fragments, which are formed by the action of a variety of peptidases. This phenomenon appears to represent an important regulatory mechanism that modulates many neuropeptide systems but is generally not acknowledged.

Animals↗

PubMed2002

Biosynthesis of triacylglycerol molecular species in an oleaginous fungus, Mortierella ramanniana var. angulispora.

The incorporation of radiolabeled lipid precursors into triacylglycerol (TG) molecular species in Mortierella ramanniana var. angulispora, an oleaginous fungus, was studied to determine the biosynthetic pathways for TG molecular species. Radiolabeled TG molecular species were separated and quantified by reverse-phase high performance liquid chromatography with a radioisotope detector. The major TG molecular species labeled by [1-(14)C]oleic acid at 30 degrees C were OOP, OOO, and OPP (TG molecular species designations represent three constituent acyl groups. G, gamma-linolenic acid; L, linoleic acid; O, oleic acid; S, stearic acid; P, palmitic acid), which were abundant TG molecular species in this fungus. The incorporation of [1-(14)C]oleic acid at 15 degrees C into these molecular species was the same, while that into most other species was decreased, suggesting that biosynthesis of major molecular species such as OOP, OOO, and OPP differs from that of other TG molecular species. [1-(14)C]Linoleic acid incorporation indicated that the major labeled molecular species were LOP and LOO, which may be due to acylation of oleoyl, palmitoyl-glycerol, or dioleoyl-glycerol by exogenous linoleic acid. This is basically the same mechanism as for OOP and OOO biosynthesis from exogenous oleic acid. [(14)C(U)]Glycerol incorporation suggested that TG molecular species containing palmitic acid such as OPP were more readily synthesized through the de novo pathway. Further experiments involving inhibitors such as sodium azide and cerulenin suggested that OOO biosynthesis included a mechanism differing from that in the cases of OOP and OPP. Trifluoperazine, which inhibits the conversion from phosphatidic acid to TG, decreased [1-(14)C]oleic acid incorporation into all molecular species, suggesting that the incorporation into all molecular species included the de novo TG biosynthetic pathway via phosphatidic acid. These results revealed that the biosynthetic pathways for TG molecular species can be classified into several groups, which exhibit different sensitivities to low temperature and inhibitors of lipid metabolism. This implies that the composition of TG molecular species is regulated through different biosynthetic pathways responsible for specific TG molecular species, providing a new insight into the biosynthesis of TG molecular species.

Carbon Radioisotopes↗

PubMed2000

Biosynthesis of L-p-hydroxyphenylglycine, a non-proteinogenic amino acid constituent of peptide antibiotics.

BACKGROUND: The non-proteinogenic amino acid p-hydroxyphenylglycine is a crucial component of certain peptidic natural products synthesized by a non-ribosomal peptide synthetase mechanism. In particular, for the vancomycin group of antibiotics p-hydroxyphenylglycine plays a structural role in formation of the rigid conformation of the central heptapeptide aglycone in addition to being the site of glycosylation. Initial labeling studies suggested tyrosine was a precursor of p-hydroxyphenylglycine but the specific steps in p-hydroxyphenylglycine biosynthesis remained unknown. Recently, the sequencing of the chloroeremomycin gene cluster from Amycolatopsis orientalis gave new insights into the biosynthetic pathway and allowed for the prediction of a four enzyme pathway leading to L-p-hydroxyphenylglycine from the common metabolite prephenate. RESULTS: We have characterized three of the four proposed enzymes of the L-p-hydroxyphenylglycine biosynthetic pathway. The three enzymes are encoded by open reading frames (ORFs) 21, 22 and 17 (ORF21: [PCZA361.1, O52791, CAA11761]; ORF22: [PCZA361. 2, O52792, CAA11762]; ORF17: [PCZA361.25, O52815, CAA11790]), of the chloroeremomycin biosynthetic gene cluster and we show they have p-hydroxymandelate synthase, p-hydroxymandelate oxidase and L-p-hydroxyphenylglycine transaminase activities, respectively. CONCLUSIONS: The L-p-hydroxyphenylglycine biosynthetic pathway shown here is proposed to be the paradigm for how this non-proteinogenic amino acid is synthesized by microorganisms incorporating it into peptidic natural products. This conclusion is supported by the finding of homologs for the four L-p-hydroxyphenylpyruvate biosynthetic enzymes in four organisms known to synthesize peptidic natural products that contain p-hydroxyphenylglycine. Three of the enzymes are proposed to function in a cyclic manner in vivo with L-tyrosine being both the amino donor for L-p-hydroxyphenylglycine and a source of p-hydroxyphenylpyruvate, an intermediate in the biosynthetic pathway.

Actinomycetales↗

PubMed2000

Structure alteration of polyketides by recombinant DNA technology in producer organisms--prospects for the generation of novel pharmaceutical drugs.

Actinomycetes are gram-positive bacteria and commercially important microorganisms. They are producers of approximately two thirds of all bioactive compounds known and they produce a great variety of compounds which have clinical application on the basis of their activity against different kinds of organisms and cells as antibacterial (macrolides, avermectins), antitumor (anthracyclines, angucyclines, aureolic acid group) and also compounds showing immunosuppresant activity (rapamycin, FK506). Most of these clinically useful pharmaceuticals produced by actinomycetes belong to the polyketide family. Polyketides comprise a wide family of chemically diverse compounds, many of which have shown bioactivity. The development of recombinant DNA technology has opened a new and exciting field of research for the generation of new bioactive compounds through genetic manipulation of the biosynthetic pathways. Researchers in this area are trying to take advantage of the enormous capability of actinomycetes to produce pharmaceutically useful compounds in order to manipulate the different biosynthetic pathways and subsequently generate novel drugs. Combinatorial biosynthesis is now emerging as a powerful tool to generate novel families of compounds by interchanging secondary metabolism genes between bioactive producing actinomycetes. Novel compounds will be the consequence of the concerted action of enzymes from different, but related, biosynthetic pathways. Insertional inactivation of selected genes and tailoring modification may also produce novel compounds that can be useful pharmaceuticals or lead compounds for further chemical modification. This minireview will present the state of the art in this field showing the different polyketides biosynthetic pathways so far characterized and how the identified genes are being used to generate structural biodiversity. Emphasis will be made on the polyketide family including type I and type II polyketides.

Actinomycetales↗

PubMed1989

The acute effects of glucose on the insulin biosynthetic-secretory pathway in a simian virus 40-transformed hamster pancreatic islet beta-cell line.

The acute regulatory effects of glucose on proinsulin synthesis at the translational level and the subsequent processing of newly made hormone through the insulin biosynthetic-secretory pathway in a clonal cell line (HIT-T15 cells) of simian virus 40-transformed hamster pancreatic islet beta-cells were examined. Proinsulin synthesis in HIT-T15 cells, assayed by immunoprecipitation, comprised 2.5-5% of the total protein synthesis during a 40-min pulse label period, though preproinsulin mRNA levels were only 0.25-0.29% of the total mRNA. There was no specific enhancement of proinsulin synthesis in response to increasing concentrations of glucose or other tested metabolites or by agents stimulating the cAMP or protein kinase-C second messenger systems during a 40-min pulse label period. Compared to that in normal beta-cells, processing of proinsulin to insulin was markedly retarded. Unlike that in normal beta-cells, newly synthesized hormone in HIT cells was secreted at a very low rate that was not enhanced by a high glucose concentration and was not preferentially secreted compared with preformed hormone. Thus, although a glucose sensor was present in HIT cells, since secretion of preformed insulin stores was stimulated 8-fold between glucose concentrations of 0 and 11 mM, regulation at several stages in the passage of newly made insulin through the biosynthetic-secretory pathway did not display the acute glucose sensitivity seen in normal nontransformed beta-cells.

Animals↗

PubMed2002

Evaluation of activity of erythrocyte pyrimidine 5'-nucleotidase (P5N) in lead exposed workers: with focus on the effect on hemoglobin.

Anemia that accompanies lead poisoning is in part the result of various inhibitory effects of lead on heme biosynthesis. Lead also increases the rate of red blood cell destruction due to the profoundly depressed activities of erythrocyte pyrimidine 5'-nucleotidase (P5N) activities. We studied parameters of the two metabolic pathways in the workers exposed to lead to evaluate P5N in the lead exposed workers and which pathway has an effect on hemoglobin (Hb) level. 29 male workers in the secondary lead smelting as high exposure group, 46 male workers in the manufacturer of inorganic pigment as low exposure group and 56 clerical male workers from another plant as non-exposed group were studied. Activity of P5N, lead concentration in whole blood (PbB), zinc protoporphyrin (ZPP), Hb, and ferritin were determined. In the present study, P5N activity of nucleotide metabolic pathway correlated with Hb after controlling indices of iron deficiency anemia (ferritin) occurring concurrently and heme biosynthetic pathway (ZPP) in the high exposure group while heme biosynthetic pathway did not correlate with Hb after controlling other two variables in exposure groups. These findings suggest that P5N rather than heme biosynthetic pathway has a major effect on Hb level even in workers without manifest hemolytic anemia.

5'-Nucleotidase↗

PubMed1992

Human keratinocytes and A-431 cells synthesize and secrete factor B, the major zymogen protease of the alternative complement pathway.

Biosynthetic radiolabeling studies demonstrate that human keratinocytes and A-431 cells, a human epidermoid carcinoma cell line, synthesize and secrete factor B as a monomeric 105-kD protein. Epithelial cell-derived factor B comigrates in SDS-PAGE with that produced by HepG2 cells, a human hepatoma cell line traditionally employed in studies of complement component biosynthesis. Comparative pulse-chase studies in A-431 and HepG2 cells show that this alternative pathway complement component is produced as co-migrating 100-kD intracellular proteins that are processed in both cell types to 105-kD extracellular factor B. Quantitatively, immunoprecipitable factor B accounts for 0.05% of radiolabeled proteins in A-431 cell culture media. Treatment of biosynthetically radiolabeled A-431 cell culture media with cobra venom factor and factor D for 60 min at 37 degrees C produces the specific factor B cleavage products Ba and Bb. These fragments are not identifiable in control culture media subjected to similar treatment in the absence of alternative pathway activators. Northern blot analysis of total cellular RNA from human keratinocytes, A-431 cells, and HepG2 cells reveals qualitative identity of a 2.8-kb factor B mRNA species in these three cell types. The relative level of factor B mRNA expression in these cells parallels their level of factor B protein synthesis (i.e., HepG2 cells greater than A-431 cells greater than human keratinocytes). Epithelial cell-derived factor B may play an important role in local inflammatory reactions and also directly interact with epithelial cell derived C3--a key classical and alternative pathway complement component recently shown to be produced by human keratinocytes.

Animals↗

PubMed1983

Flux through the de novo pyrimidine pathway in vivo. Effect of N-phosphonacetyl-L-aspartate, a potent inhibitor of aspartate transcarbamylase.

The activity of the de novo pyrimidine biosynthetic pathway has been measured in resistant and sensitive murine tumors in vivo following a single intraperitoneal dose of N-phosphonacetyl-L-aspartate (PALA) (400 mg/kg). For these studies, we utilized a gas chromatograph-mass spectrometric technique which enabled measurement of 13C incorporation from 13CO2 into the uracil nucleotide pool (sigma uracil) of tumors in situ. Flux through the de novo pathway was 75-85% inhibited 1 h after PALA treatment in both sensitive (Lewis lung carcinoma) and the resistant (L1210) tumors, but there was a lag time before this inhibition was reflected in reduced sigma uracil pools. The activity of the pathway in the Lewis lung carcinoma tumors remained maximally depressed (5-15% of control activity) for up to 48 h after the dose of PALA. In contrast, flux through the pathway of L1210 tumors remained 80% inhibited for up to 4 h following PALA administration, but recovered to 70% of control activity between 4 and 12 h after PALA treatment. Recovery of the remaining 30% of control activity in the L1210 tumor was at a much slower rate requiring between 12 and 48 h after PALA treatment to regain full activity of the pathway. This recovery of flux through the de novo pyrimidine biosynthetic pathway did not correlate with the measurement of recovery of aspartate transcarbamylase activity in similarly treated tumors. These data argue strongly in favor of the importance of the de novo biosynthetic pathway, rather than salvage mechanisms, for determining in vivo sensitivity or resistance of these tumors to PALA treatment.

Animals↗